Prebiotic and preservative uses of oil-emulsified probiotic encapsulations

ABSTRACT

Probiotic compositions derived from  Lactobacillus casei  ATCC number PTA 3945 are disclosed. More specifically, probiotic compositions containing prebiotic compositions including edible oils are provided. The probiotic/prebiotic compositions disclosed herein are packaged using a nitrogen purge instant bonding anaerobic encapsulation process to maintain high viability for extended periods.

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisionalpatent application serial No. 60/414,083 filed Nov. 26, 2002, nowabandoned, and is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 10/021,871, filed Dec. 18, 2000, the entirecontents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention is directed at probiotic compositions andmethods for making same. More specifically the present invention isdirected at probiotic compositions derived from lactic acid bacteriathat have been emulsified in prebiotic edible oils and packaged in ananaerobic encapsulation system.

BACKGROUND OF THE INVENTION

[0003] Probiotics are microbial-based dietary adjuvants thatbeneficially affect the host physiology by modulating mucosal andsystemic immunity, as well as improving nutritional and microbialbalance in the intestinal tract [Naidu A S, Bidlack W R, Clemens R A(1999) Probiotic spectra of lactic acid bacteria (LAB). Crit. Rev. FoodSci. Nutr. 39:3-126]. Lactic acid bacteria (LAB) are indigenousprobiotic microflora of mammalian gastrointestinal tract that play animportant role in the host microecology and have been credited with animpressive list of therapeutic and prophylactic properties [Naidu A S,Clemens R A (2000) Probiotics, p.431-462. In A. S. Naidu (ed.), NaturalFood Antimicrobial Systems. CRC Press, Boca Raton, Fla.]. Thesetherapeutic and prophylactic properties include, but not limited to themaintenance of microbial ecology of the gut, physiological,immuno-modulatory and antimicrobial effects [Gibson G R, Saavedra J M,Macfarlane S, Macfarlane G T (1997) Probiotics and intestinalinfections, p.10-39. In R. Fuller (ed.), Probiotics 2: Applications andpractical aspects. Chapman and Hall, London, UK]. Other LAB associatedattributes include enzyme release into the intestinal lumen that actsynergistic with LAB adhesion to alleviate symptoms of intestinalmalabsorption. Furthermore, the LAB-released enzymes help regulateintestinal pH that results in increased aromatic amino acid degradation[Mitsuoka T (1984) Taxonomy and ecology of bifidobacteria.Bifidobacteria Microflora 3:11]. LAB have also demonstrated the abilityto significantly reduce sulfide and ammonia containing compounds inanimal fecal waste and thus reduce the odor and toxicity associated withanimal excrements [Naidu A S, Xie X, Leumer D A, Harrison S, Burrill MJ, Fonda E A (2002) Reduction of sulfide, ammonia compounds and adhesionproperties of Lactobacillus casei strain KE99 in vitro. Curr. Microbiol.44:196-205].

[0004] However, the greatest potential for LAB to improve life qualityfor man and domestic animals lies in their in vivo probioticapplications. In order for LAB to exhibit beneficial probiotic effectsin vivo, the organisms must survive for extended time periods in thegut. Therefore, it is critical that probiotic LAB strains be selectedthat possess qualities that prevent their rapid removal by gutcontraction [Havenaar R, Brink B, Huis in't Veld JHJ (1992) Selection ofstrains for probiotic use, p.209-224. In R. Fuller (ed.), Probiotics,the scientific basis. Chapman and Hall, London, UK]. Effective probioticbacteria should be able to survive gastric conditions and colonize theintestine, at least temporarily, by adhering to the intestinal epithelia[Conway P (1996) Selection criteria for probiotic microorganisms. AsiaPacific J. Clin. Nutr 5:10-14].

[0005] Furthermore, in addition to increasing in vivo viability andgastrointestinal tract life span, prolonged shelf life at roomtemperature remains a commercial challenge. Lactic acid bacilligenerally require an effective delivery system that retainsprobio-functional activities (i.e. gut adhesion/retention, production ofbacteriocins/enzymes) after their revival [Salminen S, Isolauri E,Salminen E (1996) Clinical uses of probiotics for stabilizing the gutmucosal barrier: successful strains and future challenges. Antonie VanLeeuwenhoek 70:347-3581.] Though freeze-drying is an effective processfor preservation and delivery of probiotics, several physico-chemicalfactors such as humidity, aeration (oxygen availability) and temperaturecould compromise the cell viability, thereby the shelf life.

[0006] One potential additive class that may increase both in vivo lifespan and storage shelf-life is prebiotics. Prebiotics arenon-digestible, or partially digestible, food ingredients thatbeneficially affect the host by selectively simulating the growth and/oractivity of one or a limited number of bacterial species and thus ineffect improve host health. [Gibson G R, Roberfroid M B (1995) Dietarymodulation of the human colonic microbiota: Introducing the concept ofprebiotics. J. Nutr. 125:1401-1412]. Intake of prebiotics canbeneficially modulate probiotic LAB. Non-digestible oligosaccharidessuch as dietary fiber in general, and fructo-oligosaccharides (FOS) inparticular, are well known prebiotics [Roberfroid M B (1997) Healthbenefits of non-digestible oligosaccharides. Adv. Exp. Med. Biol.427:211-219]. By combining the rationale of probiotics and prebiotics,the concept of ‘synbiotics’ is proposed to characterize some colonicfoods with interesting nutritional properties in combination withhealth-enhancing functional food ingredients [Fuller R, Gibson G R(1997) Modification of the intestinal microflora using probiotics andprebiotics. Scand. J. Gastroenterol. Suppl. 222:28-31].

[0007] Essential oils are known as biological preservatives due to theirlow water activity and limited air diffusion. Several essential oilsalso known to provide various nutraceutical benefits includingantioxidant, antimicrobial, antitumor, and immune-modulatory activities.However, the prebiotic effects of essential oils on probiotic LAB areheretofore unknown. Therefore, there remains a need to enhance probioticactivity, in vivo viability and shelf life of probiotic compositionsincluding LAB. One potential solution is the application of prebioticsin combination with advanced packaging methods.

SUMMARY OF THE INVENTION

[0008] It is an object of the present invention to provide enhancedprobiotic compositions having increased viability, in vivo life span andincreased shelf life.

[0009] It is another object of the present invention to provide aprebiotic composition in combination with a new strain of lactobacillushaving increased viability, in vivo life span and increased shelf life.

[0010] It is yet another object of the present invention to preserve theprobiotic qualities of Lactobacillus casei KE01 including, but are notlimited the use of the prebiotic oil compositions of the presentinvention in combination with by an encapsulation process using a novelNitrogen-Purge, Instant-Bonding (NPIB) system.

[0011] The present invention fulfills these and other objects byproviding a new strain of Lactobacillus casei designated KE01 thatpossesses scientifically proven probiotic properties includingdemonstrated in vivo anti-enteric pathogen activity. Moreover, thepresent invention provides dietary supplements and pharmaceuticalpreparations composed of L. casei strain KE01 that are formulated in aprebiotic composition of edible oils that provided long term protectionto the organism and help maintain its proven probiotic properties andincreased in vivo life span and shelf life.

[0012] There is a need for new probiotic formulations that can be usedto treat and prevent enteric-pathogen infections and help maintain thehealth and vitality of humans and livestock. Recently, the Federal Foodand Drug Administration (FDA) has intensified its campaign against theover prescription and clinical abuse of antibiotics. The excessive useof antibiotics has increased in the number of human and animal pathogensthat are resistant to first-line antibiotics resulting in an increase ininfections that do not respond to conventional antimicrobial therapies.Moreover, the prophylactic use of antibiotics in animal feed hasresulted in an alarming increase in livestock intestinal infectionsresulting in diminished herd size and animal weight due to nutrientmalabsorption. Consequently, the number of healthy animals suitable forhuman consumption has dropped, and those that do survive long enough toreach market have significantly lower weights and consequently reducedmeat quality.

[0013] One means of preventing the rapid spread of drug resistantenteric pathogens in humans and livestock is to significantly reduceantibiotic use. However, the spread of communicable diseases includingenteric infections is inevitable due to over crowding of farms andcities. Consequently, before prophylactic antibiotic use can becompletely discontinued a suitable antimicrobial alternative must beavailable. Recent studies have indicated that the use of foodstuffs anddietary supplements containing specific strains of probioticmicroorganisms can help prevent, and in many cases actually cure,enteric pathogen diseases. However, many of the probiotic formulascurrently marketed rely on organisms including Lactobacillus spp andBifidobacteria sp (and other genera) that have not been subjected toscientific scrutiny using approved methods for assessing probioticefficacy. Consequently, too many of the “probiotic” formulas currentlyavailable lack proven in vivo anti-enteric pathogen activity. Moreover,many of the clinically effective probiotic formulations commerciallyavailable are not stable upon storage and therefore do not delivereffective amounts of viable probiotic bacteria to the user. The presentinventors have tested many commercially available preparations and foundmicrobial viability well below stated concentrations and in many casesthe present inventor has found that these commercial preparations didnot contain any viable bacteria.

[0014] The present inventors have developed methods for preparing andpackaging a new strain of L. casei, designated KE01. This new strain ofL. casei was originally from a traditional fermented yogurt-like Asiandairy product by the present inventor. Subsequently, the presentinventor characterized the isolate and the strain deposited with theAmerican Type Culture Collection (ATCC, MD, USA)). Lactobacillus caseistrain KE01 has been given the ATCC depository number PTA 3945.Moreover, the present inventors have developed preparations andpackaging systems that maintain L. casei KE01 viability such that aclinically effective dose of viable probiotic microorganisms reaches thehost.

[0015] The present invention provides a L. casei strain (KE01) thatinterferes with bacterial adherence (microbial interference) of entericpathogens such as, but not limited to enteropathogenic andenterotoxigenic E. coli, Helicobacter pylori, Campylobacter jejuni, S.typhimurium, and S. enteritidis to a variety of mammalian cell types.Moreover, the Lactobacillus of the present invention can alsocompetitively exclude (competitive exclusion) these, and other bacterialpathogens, from binding to many mammalian cells. The beneficialproperties associated with the novel Lactobacillus strain of the presentinvention have resulted in improved probiotic dietary supplements thatsupport general human and animal health. Moreover, the present inventioncan be used to provide prophylactics, therapeutics and palliatives(collectively referred to herein as “probiotics”) for conditions suchas, but not limited to, traveler's diarrhea, gastrointestinalinfections, hemolytic uremic syndrome, and gastric ulcers.

[0016] Additional novel features and qualities of this new L. caseistrain KE01 include, but are not limited to, L. casei KE01's ability toreduce sulfide concentrations by a factor exceeding 300 ppm within 48hours when exposed to a growth medium containing approximately 2000 ppmof sulfides and the demonstration of avid binding to sub-epithelialmatrices including Bio-coat™ (Collagen type-I, Collagen type IV,laminin, and fibronectin), Matrigel™ and Caco-2 cell monolayer. Mostimportantly, a reconstituted, freeze-dried preparation of the L. caseiof the present invention has been shown to effectively detachcollagen-adherent E. coli.

[0017] The methods used to maintain the viability of the L. casei of thepresent invention and preserve probiotic qualities include, but are notlimited the use of the prebiotic oil compositions of the presentinvention in combination with by an anaerobic encapsulation processusing a novel Nitrogen-Purge, Instant-Bonding (NPIB) system.

[0018] These and other beneficial probiotic properties of the new strainof Lactobacillus will be further evident by the following, non-limiting,detailed description of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 depicts the genomic fingerprint of Lactobacillus caseistrain KE01 on 1% agarose gel compared to 12 different Lactobacillustype strains based on Randomly Amplified Polymorphic DNA (RAPD) assay.

[0020]FIG. 2 depicts the phylogenic dendogram deduced from genomicfingerprinting and the relatedness of Lactobacillus strain KE01withother species of Lactobacillus type strains.

[0021]FIG. 3 depicts the standardized real-time PCR used to quantifylactobaciilus content of the present invention using KE01 specificprimers.

[0022]FIG. 4. depicts sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS_PAGE) results using the KE01 primers of the presentinvention.

[0023]FIG. 5 depicts the process for making the anaerobic encapsulationsystem using NPIB in accordance with the teachings of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Lactic acid bacteria (LAB) are indigenous microflora of mammaliangastrointestinal tract that play an important role in the hostmicroecology and have been credited with an impressive list oftherapeutic and prophylactic properties. These therapeutic andprophylactic properties include, but not limited to the maintenance ofmicrobial ecology of the gut, physiological, immuno-modulatory andantimicrobial effects. Other LAB associated attributes include enzymerelease into the intestinal lumen that act synergistically with LABadhesion to alleviate symptoms of intestinal malabsorption. Furthermore,the LAB enzymes help regulate intestinal pH which results in increasedaromatic amino acid degradation. [Fuller, R. Probiotic foods—current useand future developments. IFI NR 3:23-26 (1993); Mitsuoka, T. Taxonomyand ecology of Bifidobacteria. Bifidobacteria Microflora 3:11 (1984);Gibson, G. R. et al., Probiotics and intestinal infections, p.10-39. InR. Fuller (ed.), Probiotics 2: Applications and practical aspects.Chapman and Hall, London, U.K. (1997); Naidu A S, et al., Probioticspectra of lactic acid bacteria (LAB). Crit Rev Food Sci Nutr 39:3-126(1999); Naidu, A. S., Clemens, R. A. Probiotics, p.431-462. In A. S.Naidu (ed.), Natural Food Antimicrobial Systems. CRC Press, Boca Raton,Fla. (2000)]

[0025] Lactic acid bacteria have also demonstrated the ability tosignificantly reduce sulfide and ammonia containing compounds in animalfecal waste and thus reduce the odor and toxicity associated with animalexcrements. This ex vivo LAB application is becoming increasingly moreimportant as agro-businesses expand and as communities continue theirseemingly never ending encroachment into previously unoccupied ruralareas. For example, LAB has been demonstrated to eliminate offensiveodors and reduce hydrogen sulfide production associated with hatcherywaste when cockerel chicks and shell waste are blended with a mixturecontaining 15% carbohydrate and LAB. Moreover, LAB compositions havedemonstrated efficacy in diminishing the Escherichia coli and Salmonellacontent of hatchery waste to negligible levels.

[0026] Additionally, the odor and viscosity of poultry offals such asbroiler-processing waste is significantly reduced by L. acidophilusmediated lactic acid fermentation. Furthermore, preparations containingLAB have been reported to accelerate the breakdown of hard-to-degradecarbohydrates and decrease the ammonia production by porcine cecalbacteria. Finally, ex vivo L. casei FG1 and L. plantarum silagefermentation significantly reduces ammonia levels by inhibitingurea-splitting organisms. [Deshmukh, A. C., Patterson, P. H.Preservation of hatchery wastes by lactic acid fermentation. 1.Laboratory scale fermentation. Poult Sci 76:1212-1219 (1997);. Russell,S. M. et al., Lactic acid fermentation of broiler processing waste:physical properties and chemical analyses. Poult Sci 71:765-770 (1992);Tibbetts, G. W. et al., Poultry offal ensiled with Lactobacillusacidophilus for growing and finishing swine diets. J Anim Sci 64:182-190(1987); Sakata, T. et al., Probiotic preparations dose-dependentlyincrease net production rates of organic acids and decrease that ofammonia by pig cecal bacteria in batch culture. Dig Dis Sci 44:1485-1493(1999); Cai, Y. et al., Effect of applying lactic acid bacteria isolatedfrom forage crops on fermentation characteristics, aerobic deteriorationof silage. J Dairy Sci 82:520-526 (1999); Modler, H. W. et al.,Bifidobacteria and bifidogenic factors. Can Inst Food Sci Tech 23:29-41(1990)].

[0027] However, the greatest potential for LAB to improve life qualityfor man and domestic animals lies in LAB in vivo probiotic applications.In order for LAB to exhibit beneficial probiotic effects in vivo, theorganisms must survive for extended time periods in the gastrointestinaltract. Therefore, it is critical that probiotic LAB strains be selectedthat possess qualities that prevent their rapid removal by gutcontraction. Effective probiotic bacteria must able to survive gastricconditions and colonize the intestine, at least temporarily, by adheringto the intestinal epithelium. Consequently, LAB that demonstrate anenhanced ability to adhere to mucosal surfaces, and therefore possessimproved bacterial maintenance and prolonged gastrointestinal tractresidence times, have a competitive advantage over LAB that do not.[Salminen, S. et al., Clinical uses of probiotics for stabilizing thegut mucosal barrier: successful strains and future challenges. AntonieVan Leeuwenhoek 70:347-358 (1996); Conway, P. Selection criteria forprobiotic microorganisms. Asia Pacific J Clin Nutr 5:10-14 (1996);Havenaar, R. et al., Selection of strains for probiotic use, p.209-224.In R. Fuller (ed.), Probiotics, the scientific basis. Chapman and Hall,London, U.K. (1992)].

[0028]Lactobacillus can successfully colonize the mammaliangastrointestinal tract through a number of different mechanisms. Forexample, some bacterial species bind to various sub-epithelial matrixproteins and specific receptors on the intestinal mucosa. Other speciesmay adhere to mammalian intestinal cells via mechanisms that involvedifferent combinations of carbohydrate and protein factors on thebacteria and host eukaryotic cell surfaces. However, regardless of themechanism(s) of attachment, it is the ability of LAB to successfullycolonize the human gastrointestinal tract that provides LAB withprobiotic qualities. [Greene, J. D., Klaenhammer, T. R. Factors involvedin adherence of lactobacilli to human Caco-2 cells. Appl EnvironMicrobiol 60:4487-4494 (1994); Sarem, F. et al., Comparison of theadherence of three Lactobacillus strains to Caco-2 and Int-407 humanintestinal cell lines. Lett Appl Microbiol 22:439-442 (1996); Naidu, A.S., et al., Particle agglutination assays for rapid detection offibronectin, fibrinogen, and collagen receptors on Staphylococcusaureus. J Clin Microbiol 26:1549-1554 (1988); Wadstrom, T. et al.,Surface properties of lactobacilli isolated from the small intestines ofpigs. J Appl Bacteriol 62:513-520 (1987); Bernet, M. F. et al.,Lactobacillus acidophilus LA 1 binds to cultured human intestinal celllines and inhibits cell attachment, invasion by entero-virulentbacteria. Gut 35:483-489 (1994); Jin, L. Z. et al., Effect of adherentLactobacillus spp. on in vitro adherence of salmonellae to theintestinal epithelial cells of chicken. J Appl Bacteriol 81:201-206(1996); Reid, G. et al., Influence of lactobacilli on the adhesion ofStaphylococcus aureus and Candida albicans to fibers and epithelialcells. J Ind Microbiol 15:248-253 (1995)].

[0029] Generally speaking probiotic bacteria exert their beneficialeffects by displacing invasive or toxigenic pathogenic enteric bacteria(enteric pathogens) from the intestinal mucosa through a competitivebinding process. Enteric pathogens such as, but not limited to,enteropathogenic Escherichia coli (EPEC), enterotoxigeneic E. coli(ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeriamonocytogenes must be able to successively colonize an animal'sintestinal tract in order to cause disease.

[0030] Probiotic compositions exert optimal beneficial qualities whenthe percentage of viable probiotic bacteria is high. However,maintaining bacteria viability constitutes a significant challenge tothe probiotic industry. Consequently, most probiotic compositions have arelatively short shelf life or are used with percent viabilities thatare suboptimal. Many probiotic compositions, including those of thepresent invention are made using selected stains of (or combination of)bacteria including Lactobacillus acidophilus, L. amylovorus, L. brevis,L. bulgaricus, L. casei spp. casei, L. casei spp. rhamnosus, L.crispatus, L. delbrueckii ssp. lactis, L. fermentum, L. helvaticus, L.johnsonii, L. paracasei, L. pentosus, L. plantarum, L. reuteri, and L.sake: the genus Bifidobacterium including: B. animalis, B. bifidum, B.breve, B. infantis, and B. longum: the genus Pediococcus including: P.acidilactici: the genus Propionibacterium including: P. acidipropionici,P. freudenreichii, P. jensenii, and P. theonii: and the genusStreptococcus including: S. cremoris, S. lactis, and S. thermophilus(collectively referred to herein after as lactic acid bacteria, or LAB).

[0031] Presently, probiotic compositions are produced using cultured,concentrated LAB that are dried or lyophilized and then mixed withstabilizing ingredients such proteins and sugars including, but notlimited raffinose, soybean oligosaccharides, fructooligosaccharides,galactooligosaccharides, galactosyl lactose and palatinose, lactulose,lactitol, xylitol, sorbitol, mannitol, trehalose, glucose, sucrose,fructose, maltose, milk, milk powders, whey, whey protein concentrates,casein, casein hydrolysates, lactoferrin, lactoperoxidase,lactoglobulins, glycomacropeptides, lacto-saccharides, and lacto-lipids.These probiotic mixtures are milled into small granule or fine powdersand then sealed in various pharmaceutically acceptable forms andpackaged. Alternatively, liquid preparations are provided that must bestored at refrigerator temperatures. However, regardless of the form,whether dried or liquid, LAB viability begins to drop dramatically soonafter shipping to retail outlets and end users.

[0032] The present inventors have surprisingly discovered that certainedible oils not only protect LAB viability better than prior artstabilizing ingredients such proteins and sugars, actually enhanceviability resulting in a prebiotic effect. As previously discussed,prebiotics are non- or partially digestible food ingredients thatbeneficially affect the host by selectively simulating the growth and/oractivity of one or a limited number of bacterial species in the colon,and thus in effect improve host health. One embodiment of the presentinvention is a dietary supplement comprising approximately from 10⁵ to10¹¹ colony forming units of viable LAB per mL of prebiotic edible oil.However, it is understood that the probiotic compositions of the presentinvention are not limited by this range and may in fact comprises fromapproximately 1 to >10¹² colony forming units of viable lactobacilli permL of prebiotic edible oil.

[0033] In one embodiment of the present invention the LAB Lactobacilluscasei strain KE01 having the American Type Culture Collection (ATCC)accession number PTA 3945. In one embodiment of the present inventionthe prebiotic/probiotic composition comprises a hard two piece capsulefilled with a lactobacillus composition suspended in at least one edibleoil. In this embodiment the hard two piece capsule has been purged withan inert gas upon filling and sealed to assure an oxygen-freeenvironment, as described more fully below. The hard two piece capsulecan be derived from either vegetables, animal gelatin or synthetic andnatural polymers.

[0034] Throughout this specification the present invention may bereferred to as a probiotic composition, a lactobacillus containingcomposition, a dietary supplement, or a probiotic/prebiotic composition.All of these aforementioned terms mean a composition, regardless of formor the presence or absence of other ingredients, that contains viableand or/ non-viable LAB and at least one prebiotic, edible oil. In oneembodiment the LAB is Lactobacillus casei strain KE01 having ATCCaccession number PTA 3945 or it genetic equivalent as determined usingthe methods detailed herein.

[0035] In one embodiment of the present invention an animal is providedwith a single dose containing from approximately 10⁵ to 10¹¹lactobacilli per gram of probiotic composition. The total amountconsumed will depend on the individual needs of the animal and theweight and size of the animal. The preferred dosage for any givenapplication can be easily determined by titration. Titration isaccomplished by preparing a series of standard weight doses eachcontaining from approximately 10⁵ to 10¹¹ lactobacilli per gram. Aseries of doses are administered beginning at 0.5 grams and continuingup to a logical endpoint determined by the size of the animal and thedose form. The appropriate dose is reached when the minimal amount oflactobacilli composition required to achieve the desired results isadministered. The appropriate dose is also known to those skilled in theart as an “effective amount” of the probiotic compositions of thepresent invention.

[0036] For example, if it is desired to reduce the symptoms associatedwith irritable bowel syndrome in an animal, one measured dose asdescribed above is administered daily, escalating the dose eachsuccessive day in 0.5 grams increments until symptoms subside. In oneembodiment of the present invention the preferred dose is betweenapproximately 10³ to 10⁸ viable lactobacilli per kilogram of body weight(the weight of the animal recipient) per day. This equates toapproximately 10 billion viable LAB per day for the average healthyadult human. By extrapolation, it is a simple matter to calculate theapproximate dose appropriate for any animal of any weight. It isunderstood that this is a non-limiting example that can be varied asappropriate by persons having skill in the art of prescribing probioticcompositions or by using the titration method provided above.

[0037] The probiotic compositions of the present invention can beadministered to any animal in need of thereof including, but not limitedto mammals, birds, reptiles and fish. Typical applications includeadministering the probiotic compositions of the present invention tohumans, horses, swine (pigs), cows, sheep, dogs, cats, rabbits,chickens, turkeys, pheasants, quail, parakeets, parrots, and other wildand domesticated animals.

[0038] Specifically, the probiotic compositions of the present inventioncan be used to inhibit or treat enteric pathogen-associated diseaseswhen administered to an animal in need thereof using the methodsdescribed in the present specification. Enteric pathogen diseasesinclude those diseases caused by pathogens such as diarrhea, irritablebowel syndrome and intestinal hemorrhages. Examples of enteric pathogensassociated with these diseases include, but not limited toenteropathogenic Escherichia coli (EPEC), enterotoxigeneic E. coli(ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeriamonocytogenes. It is theorized by the present inventor, and not offeredas a limitation, that the inhibition and treatment of the entericpathogen diseases is accomplished by the probiotic composition of thepresent invention through a competitive binding process. That is, theprobiotic lactobacilli of the present invention compete with entericpathogens for binding sites on the intestinal mucosa. Because theprobiotic lactobacilli of the present invention have a higher affinityand avidity for these binding sites than the enteric pathogens, theprobiotic lactobacilli of the present invention displace the entericpathogens into the intestinal milieu where they are harmlessly flushedfrom the intestines by normal metabolic processes.

[0039] In one embodiment of the present invention the probiotic organismof the present invention was isolated from a traditional fermentedyogurt-like Asian dairy product. The screening process was limited totraditional fermented yogurt-like Asian dairy products with at least aten-year history of safe human consumption. Probiotic bacteria isolationwas performed using three selective microbiological media using methodsknown to those of ordinary skill in the art of microbiology.Lactobacilli selective media included SL medium supplemented with 0.05%cystein, Bifidobacterium spp. were selected for using trypticase peptoneyeast extract medium containing antibiotics; and for Streptococcus spp.were isolated using trypticase yeast extract cystein medium.

[0040] Candidate probiotic lactobacilli were be catalase negative,glucose homofermentative, Gram-positive non-spore forming rodsdemonstrating low pH, gastric acid and bile resistance. The lactobacilliisolates' inability to grow at pH 9.0 coupled with their ability to growon acetate containing media served to distinguish them fromCarnobacterium spp. A total of 81 isolates were classified as candidateprobiotic lactobacilli based on these criteria and were furthercharacterized with respect to the following criteria: i) resistance tolow pancreatic juice; ii) adherence ability to sub-epithelial matricessuch as Biocoat™ Matrigel™ (Becton Dickinson, Bedford, Mass.) and tocultured intestinal epithelial cells (Caco-2 cell line); iii) theirability to competitively exclude enterohemorrhagic E. coli serotypeO157:H7 adherent to collagen matrices; and iv) their capacity to reduceammonia and sulfide containing compounds.

[0041] After analyzing all 81-candidate probiotic lactobacilli, twostrains were identified having all of the above-identifiedcharacteristics. These strains were designated strain KE97 and strainKE99 (re-designated KE01). Finally the growth-multiplication rate(generation time as determined by impedance detection using BioMerieux™Bactometer System), stability of strains in continuous culture,freeze-drying and revival characteristics, and aroma/flavor profileswere ascertained for each strain.

[0042] The isolated Lactobacillus casei strain KE01 organism ismaintained in a substantially pure culture for use in preparingprobiotic compositions of the present invention. As used herein“substantially pure culture” refers to a bacteriological culture thatresults in only one identifiable organism when cultured on solid orsemi-solid microbiological culture media. It is understood thatextremely low levels of cells from other bacterial species may bepresent; however, these cells are either non-viable, non-cultivable orbelow the threshold of detection using classical, non-genome-based,microbiological techniques. The term “non-genome-based” is intended toexcluded such methods as PCR detection or similar methods used to detectmicrobial DNA or RNA.

[0043] Real-time PCR (RT-PCR) for Specific Measurement of Strain KE01for Quality (Purity) and Quantity (Total Bacterial Numbers)

[0044] In addition to estimating CFU counts by standard plate counts onMRS agar, RT-PCR assay was used for qualitative/quantitative measurementof survival and revival rates of strain KE10 in oil-based formulations.Primers specific for strain KE01 were developed and the RT-PCR wasperformed as described below:

Primer Design

[0045] Primers for the amplification of species-specific Lactobacilluscasei KE01 (ATCC-PTA3945) set were synthesized (Table 1). All primerswere synthesized using the standard desalting processes (Integrated DNATechnologies Coralville, Iowa). The primer set for strain KE01 wasdesigned by amplification of the 16S rRNA gene of Lactobacillus spp.,sequenced (City of Hope Research Center Duarte, Calif.) and compared forbase additions, deletions, substitutions, etc. against a public genebank using Lasergene (DNAStar, Madison, Wis.) software. A primer set forReal-Time PCR was then developed specific for the KE01 strain. Thisprimer set creates a 154 bp fragment. All primer sets were rehydrated to100 mM with molecular grade water (Eppendorf, Hamburg, Germany) andstored at −20° C. until used. TABLE 1 Name Primer Sequence KE01 F1 (SEQID NO 1) 5′-TTG TCA CCG GCA GTT CTT AC-3′ lac upp (SEQ ID NO 2) 5′-TGTCGT CAG CTC GTG TCG T-3′

[0046] Real-Time PCR

[0047] Quantitative real-time PCR on the iCycler iQ was performed induplicate on 7 μl of template DNA per 25 μl reaction. The iQ supermixreactions consisted of iQ supermix (Bio-Rad, Hercules, Calif.) at afinal concentration of 1×, 10 nM fluorescein, SYBR Green I with thesupermix master mix (50 mM KCl, 20 mM Tris-HCl, 0.2 mM dNTP, 25 units/mliTaq DNA Polymerase, 3 mM MgCl₂).

[0048] Reactions were amplified in a 96-well thin wall PCR plate(Bio-Rad, Hercules, Calif.) using the following parameters: 95° C. for 3min, followed by 40 cycles of denaturation at 95° C. for 10 sec,annealing at 66° C. for 15 sec. Melt curve analysis was performedimmediately following amplification by ramping the temperature from 55°C. to 95° C. The presence of a single PCR product was verified both bythe presence of a single melting temperature peak representing aspecific product (vs. a nonspecific primer-dimer peak) using iCycler iQanalysis software and by detection of a single band of the expected sizeon a 12.5% TBE-polyacrylamide gel.

[0049] A standard curve was produced and used to determine theconcentrations of the samples. The concentration values were 0.16 ng,0.016 ng, 0.0016 ng, and 0.00016 ng and used from stock known bacteria(strain KE01). The standard concentration was used from stock E. coli(ATCC 43895), and strain KE01. For total lactobacilli estimations amixture of strains, i.e. L. casei (ATCC393), L. pentosus (ATCC 8041), L.plantarum (ATCC 14917), L. paracasei subsp. paracasei (ATCC 25302) andstrain KE01 were used. The DNA of all stock bacteria were extracted andthe DNA concentrations were determined by the Picogreen® Quantificationkit and diluted accordingly in 1X TE buffer to the appropriateconcentrations suitable for RT-PCR analysis. FIG. 3 depicts the resultsof real-time PCR standards for strain KE01. Each standard concentration(0.16, 0.016, 0.0016, 0.00016 and 0 ng) was run in duplicate (two linesper standard).

[0050] DNA Fingerprinting by Random Amplified Polymorphic DNA (RAPD)Assay

[0051] The RAPD protocol uses PCR for generating a unique fingerprintfor bacterial identification. The analysis by PCR can be performed in arapid and reliable manner. Accordingly, the RAPD assay has been used formolecular identification and finger printing of strain KE01. A total of12 Lactobacillus spp. type strains from the ATCC collection were fingerprinted and compared with the KE01. For the DNA fingerprinting all thelactobacillus strains were cultivated overnight in MRS broth (Difco).The Lactobacillus strains analyzed for DNA fingerprint are listed inTable 2. TABLE 2 Lactobacillus spp. SOURCE Lactobacillus strain KE01en-N-tech, Inc., California, USA Lactobacillus acidophilus Human [L 917;IFO 13951; NCIB ATCC 4356 8690] Lactobacillus amylovorus Cattlewaste-corn silage ATCC 33620 Lactobacillus brevis Human feces ATCC 14869Lactobacillus casei subsp. Cheese [IAM 12473; Orland L-323] casei ATCC393 Lactobacillus casei subsp. [BUCSAV 227; P. A. Hansen 300; rhamnosusATCC 7469 NCDO 243; NCIB 6375; NCTC 6375; NRC 488] Lactobacillusdelbrueckii subsp. Swiss cheese [DSM 20072; IAM 12476; lactis ATCC 12315NCDO 1438] Lactobacillus fermentum Fermented beets [NCIB 11840] ATCC14931 Lactobacillus helvaticus Swiss cheese ATCC 15009 Lactobacillusparacasei subsp. [NCDO 151; R094] paracasei ATCC 25302 Lactobacilluspentosus [DSM 20314; NCDO 363; NCIB 8026] ATCC 8041 Lactobacillusplantarum Pickled cabbage [IAM 124771] ATCC 14917 Lactobacillus reuteriHuman feces ATCC 23272

DNA Extraction Method

[0052] DNA was extracted from the lactobacilli using the Wizard GenomicDNA Purification Kit (Promega, Wis., USA). Briefly, 1 mL of 24-h grownMRS broth culture of each lactobacillus spp. was harvested bycentrifugation, cells were resuspended in 50 mM EDTA and treated with 10mg/mL lysozyme (Sigma, Mo., USA) at 37° C. for 60 min. Lactobacillicells were pelleted by centrifugation and supernatant was removed. Thebacterial pellets were resuspended in the nuclei lysis solution andincubated at 80° C. for 5 minutes. Cell suspension was allowed to coolto room temperature and RNAse was mixed into the solution. Thesuspension was incubated at 37° C. for 60 min. After incubation, proteinprecipitation solution was added to the mixture. Solution was mixed onvortex and incubated on ice for 5 min. The mixture was centrifuged for 3min at 15K X g, supernatant was transferred to a fresh tube and the DNAwas precipitated with isopropyl alcohol. The DNA was centrifuged and theisopropyl alcohol was aspirated. The DNA pellet was washed with 70%ethanol and harvested by centrifugation. Ethanol was removed and thepellet was allowed to dry. The DNA was resuspended in tris-EDTA buffer.

PCR Amplification of Extracted DNA

[0053] One microliter of the extracted DNA was used in the PCRreactions, which were carried out on the iCycler (Bio-Rad, CA, USA)using a single arbitrary nucleotide sequence according to Cocconcelli,et al. (1995). A 2X PCR solution-Premix Taq (Takara, Shiga, Japan) wasused for each reaction. Each reaction contained a total volume of 50 μL,1.25 units of Takara Ex Taq DNA Polymerase, 1X Buffer, 200 μM dNTP Mix(2.5 mM each). Final concentration of the primer was 4 μM, and theprimer used for the amplification was 5′-AgCAgCgTgg-3′ (OperonTechnologies, Inc., CA, USA). The reaction mixtures with the templateDNA were cycled through the following temperature profile: 1 cycle of94° C. for 5 min; 40 cycles of 94° C. for 1 min; 29° C. for 1 min; rampto 72° C. 1.5 min and held at 72° C. for 1.5 min; 1 cycle of 72° C. for2 min; and held at 4° C. [Cocconcelli, P S et al., Development of RAPDprotocol for typing of strains of lactic acid bacteria and entercocci.Lett. Appl. Microbiol. 21:376-379 (1995)].

Gel Electrophoresis

[0054] Aliquots of each RAPD amplified reaction (10 μL) were analyzed by1% (wt/vol) agarose gel electrophoresis in Tris-borate-EDTA bufferaccording to Sambrook et al. (1989). Gels were run for 2 hr at 120Vwithout cooling. The DNA molecular weight marker Hyperladder I (Bioline,Randolph, Mass., USA) was used as the standard. After electrophoresisthe gel was stained with ethidium bromide (5 μg/mL) for 10 min, washedfor 5 min and visualized and analysed on a Fluor-S Multilmager (BioRad,CA, USA). [Sambrook, J., Fritsch, E. F., Maniatis, T. MolecularCloning—A Laboratory Manual, 2nd Edition. Cold Spring Harbor LaboratoryPress, New York (1989)].

[0055] The RAPD assay using a single 10-mer primer produced distinctbanding patterns of variable intensities and numbers of amplifiedproducts on 1% agarose gel with DNA samples of various lactobacillusreference strains and strain KE01. Comparison of the different speciesfragments on the gel to the reference Lactobacillus spp. was noted. Thebanding pattern with documentation as depicted in FIG. 1 and TABLE 3serves to uniquely identify strain KE01 and provided a genomicfingerprinting library. Based on the genomic fingerprinting a dendogramwas deduced as shown in FIG. 2. Ward's Cluster Method of PhylogenicAnalysis was applied. This method minimizes the Sum of Squares of anytwo clusters that can be formed at each step, creating clusters of smallsizes. Based on the phylogenic analysis, strain KE01 showed TABLE 3 LaneBand Number Number Mol. Wt. (bp) Identification 2 1 4661.359 StrainKE-01 2 2 2986.357 2 3 2457.454 2 4 2255.342 2 5 1565.537 2 6 1354.07 27 991.287 2 8 904.264 2 9 596.721 3 1 2457.454 Lactobacillus acidophilus3 2 1231.998 ATCC# 4356 3 3 1003.944 3 4 904.264 3 5 861.774 3 6 617.8134 1 4721.177 Lactobacillus amylovorus 4 2 1969.14 ATCC# 33620 4 31894.057 4 4 1725.33 4 5 1672.497 4 6 1271.414 4 7 1203.24 4 8 1023.9 51 3611.082 Lactobacillus brevis 5 2 3259.978 ATCC# 14869 5 3 2534.42 5 42087.693 5 5 1864.831 5 6 1271.414 5 7 1031.994 5 8 940.583 5 9 888.5765 10 651.854 6 1 2545.999 Lactobacillus delbrueckii 6 2 1281.464 ssp.lactis ATCC# 12315 6 3 1036.064 6 4 940.583 6 5 892.472 6 6 645.528 7 17490.875 Lactobacillus fermentum 7 2 2545.999 ATCC# 14931 7 3 1732.051 74 1281.464 7 5 1036.064 7 6 953.011 7 7 900.317 7 8 655.041 8 114117.898 Lactobacillus helveticus 8 2 4936.649 ATCC# 15009 8 3 3634.2418 4 2776.318 8 5 2394.989 8 6 2105.687 8 7 1529.442 8 8 1036.064 9 13588.07 Lactobacillus paracasei 9 2 1271.414 ssp. paracasei ATCC# 9 31107.771 25302 9 4 1027.939 9 5 932.388 9 6 892.472 9 7 636.154 10 16200.546 Lactobacillus plantarum 10 2 4968.224 ATCC# 14917 10 3 4263.32610 4 1969.14 10 5 1685.552 10 6 1322.462 10 7 1231.998 10 8 1027.939 109 223.284 11 1 2592.843 Lactobacillus casei ssp. 11 2 2324.117 rhamnosusATCC#7469 11 3 1685.552 11 4 1430.79 11 5 1312.091 11 6 1212.751 11 71011.88 11 8 780.732 12 1 9035.455 Lactobacillus pentosus 12 2 6787.29ATCC# 8041 12 3 2405.288 12 4 2198.014 12 5 1453.497 12 6 1261.443 12 71198.513 12 8 982.651 12 9 667.942 13 1 2945.799 Lactobacillus casei 132 2374.522 ssp. casei ATCC# 393 13 3 2169.899 13 4 1436.433 13 51217.534 13 6 974.089 13 7 876.989 13 8 839.445 13 9 661.46 13 10583.785 14 1 2324.117 Lactobacillus reuteri 14 2 2114.743 ATCC# 23272 143 1402.903 14 4 1184.442 14 5 944.708 14 6 832.131 14 7 639.264

[0056] 13% homology with Lactobacillus helvaticus ATCC15009 and 55%homology with Lactobacillus casei ssp. rhamnosus ATCC7469.

[0057] A pure culture of Lactobacillus casei strain KE01 was depositedwith the American Type Culture Collection, Bethesda, Md., which wasassigned the number ATCC PTA 3945.

[0058] The present inventors have demonstrated that by suspending lacticacid bacilli in edible oils, probiotic/prebiotic compositions resulthaving greater viability than previously possible. However, as will beapparent from the examples that follow, merely because a particular oilis edible, and in fact may have health giving qualities of its own, itdoes not necessarily follow that all edible oils are satisfactoryprebiotics. As can be seen in Table 4 below, the percent recovery versusthe control ranged from approximately 59% to over 370% during a fourweek storage. Based on these observation, the present inventorsconcluded that fish oil, olive oil, rice-bran oil, and soybean oildemonstrate prebiotic effects where the other oils tested were eithernot prebiotic, or were in fact antibacterial.

[0059] As discussed above, the present inventors have discovered thatselecting the proper prebiotic oil provides a means for preserving andenriching the viability and hence probiotic activity of the Lactobacillipreparations of the present invention. (Note, non-viable Lactobacillimay also possess probiotic effects, however, non-viable preparations maynot benefit significantly from the prebiotic formulations of the presentinvention.) However, when the optimum prebiotic oil is combined with anoptimized delivery system, overall probiotic activity can be increasedand/or preserved over a longer time period.

[0060] Probiotic lactobacilli require anaerobic or microaerophilicconditions for optimum viability. Though oil immersion provides lowwater activity and limited oxygen diffusion, any evacuation of oxygenfrom the microenvironment would enhance the probiotic preservation.Accordingly, a novel anaerobic encapsulation system usingnitrogen-purge-instant-bonding (NPIB) as depicted in FIG. 5 has beendeveloped to protect the oil-emulsified probiotic formulations of thepresent invention. This process provides an optimal microenvironmentanaerobic/microaerophilic) condition ideal for the probiotic bacteria.As used herein, the term “anaerobic” will mean a low oxygen tensionenvironment and includes a strict anaerobic environment andmicroaerophilic environments. The NPIB system of the present inventionprovides for oxygen displacement simultaneously with sealing the oilemulsified lactobacilli in a hard, two-piece capsule.

[0061] Moreover, the NPIB system of the present invention may also beused with other compositions including, but not limited to powders,oil-based liquids, oil-based suspensions, oil-based pastes, waxes,low-water content emulsions and a variety of bioactive compoundsincluding, but not limited to lactoferrin. Moreover, the choice ofcapsular material is highly flexible. For example in one embodiment ofthe present invention natural two piece hard gelatin/vegetable capsulesare used, in another embodiment soft gelatin/vegetable capsules areused, additional capsule compositions included assorted synthetic andnatural polymers. The NPIB system of the present invention can beincorporated into any type of capsule filling equipment, including themanual, semi or fully automated as well as continuous or intermittentmotion devices.

[0062]FIG. 5 depicts the process for making the anaerobic encapsulationsystem of the present invention. After separation of cap and body (504and 506 respectively in FIGS. 5), the oil-based bioactive composition ofthe present invention is added to the capsule body 506. Next, ananaerobic gas such as but not limited nitrogen or argon is injected intothe capsule cap 504 and a sealing solution is applied to the inner,lower section of the cap (seam 502). This results in instant bonding ofthe cap 504 and body 506 at the time when cap and body are joined duringthe closing step. In one embodiment the sealing solution is anaqueous-alcohol solution comprising from approximately 50% to 99%ethanol, 90% being preferred (for vegetable capsules) or 10% to 70%isopropyl alcohol (IPA), 60% being preferred (for gelatin capsules) and15%-30% IPA being optimum for vegetable-based capsules. However, thoseskilled in the art of formulation and filling technologies understandthat other organic solvents are suitable and combinations of differentsolvents may be used. For example, and not intended as a limitation,vegetable capsules comprising plant-derived celluloses are soluble atvarying degrees in ethanol, isopropyl alcohol or water. The specificratio of these three solvents in a sealant is dependant on severalfactors such as (i) rate of sealant application, (ii) volume of sealantapplied, (iii) method of sealant application, (iv) degree of sealantatomization, (v) method of atomization, and (vi) residence time. Thesefactors are also strictly dependant on the wet-ability, solubility andsoftening properties of the capsular materials. Accordingly, thefollowing conditions should be controlled during the sealing process:(a) the softening should not exceed the structural rigidity of the capduring the closing step. Beyond a certain limit, the cap deforms andfails to hold to the body; (b) the wet-ability and solubility propertiesof the cap should be retained and effectively transferred to the body.This would allow proper fusion of the inner surface of the cap with theouter surface of the body during the closing step; (c) The cap and headshould have excellent closure compatibility, since ethanol and isopropylalcohol evaporate rapidly, while water evaporates slowly over a periodof few hours; (d) the wet-ability and solubility properties are highlycritical during ‘instant bonding’ process.

[0063] In one embodiment of the present invention the nitrogen purge andsealing steps are done while the capsule is in a vertical position FIG.5. This differs from prior art procedures that fill and close thecapsules in the vertical position and then immediately ejected thefilled capsule into the horizontal position. This changed orientation(from vertical to horizontal) causes the capsule's fluid contentsmigrate into capsule seam 502 between the capsule halves 504 and 506.Once the capsule seam is contaminated with fluid, the sealing step isconsiderably less efficient resulting in leakage of product from thecapsule and contamination of the capsule contents with air.

[0064] The NPIB system of the present invention effectively solves thisproblem by sealing the capsule immediately after filling and purgingbefore any change in capsule potion is initiated. Consequently, thecapsule seam remains uncontaminated and the seal integrityuncompromised.

[0065] In one embodiment of the present invention a method for making aprobiotic composition is provided comprising emulsifying a Lactic AcidBacteria (LAB) in an edible oil, the edible oil having prebioticproperties. Next a hard capsule body 506 is filled with the emulsifiedprobiotic while the capsule is in the vertical orientation. The filledcapsule body 506 remaining in the vertical orientation. Next the cap 504is purged with a Noble Gas such as, but not limited to Nitrogen or Argonand a sealing solution is applied to the cap seam 502. Followingapplication for the sealant to the cap seam 502, the cap 504 and capsulebody 506 are brought together and instantly bonded. In some embodimentsbone-dry carbon dioxide gas may be used as well. Finally the filled,purged capsule is sealed with an aqueous-organic solvent solution. It isunderstood by those skilled in the art of formulation and filling thatthe purging process can be done contemporaneously with the fillingprocess.

[0066] The following examples are meant to be non-limiting in theirscope. To the extent specific ranges, compositions, ingredients andconditions are called out these are to be considered merely exemplaryfor the purposes of teaching preferred embodiments and or best mode.

EXAMPLES Example 1 Preparation and Testing of Prebiotic Oils

[0067] A pure culture of Lactobacillus casei strain KE01 is revived in afermentation broth media containing proteins, vitamins, minerals andcarbohydrate source. A seed culture is prepared in a fermentor attachedto fermentation vessel. Microbial purity is monitored at defined timepoints (through log phase and end cycle) during the fermentationprocess. The microbial mass is harvested in a sanitized separator andthe slurry of cell concentrate was freeze-dried after mixing withcarriers and cryoprotectants. The freeze-dried lactobacillus cellconcentrate was milled to fine powder using sanitized milling equipment.The quality of lactobacilli powder was assured for purity and viabilityprior to use. The viability was about 10¹⁰ CFU lactobacilli/gm ofpowder.

[0068] The probiotic lactobacilli powder is then thoroughly mixed withdifferent oils listed in Table 4 to a final 0.1% emulsion (about 0.1gpowder containing 10⁹ CFU lactobacilli mixed in 1 mL oil). In oneembodiment of the present invention the emulsion is prepared in 10 mLsterile glass tubes and homogenized in a vortex blender. The blendedmixture is then and sealed with air tights caps before testing. Multipletubes for each type of oil emulsion were prepared, and the tubes werestored at room temperature (28° C.). Tubes containing 10⁹ CFUlactobacilli powder without any oil served as growth controls.

[0069] At weekly intervals for four weeks a 1 mL sample of each oilcontaining L. casei was tested for viability as follows: nine mL ofsterile deMann Rogosa Sharpe (MRS) broth was added to each test aliquotand the contents were homogenized using a vortex blender. Serialten-fold dilutions of the homogenized mixture were prepared in MRS brothtubes. All tubes were incubated at 37° C. for 18 hours. Lactobacilligrowth was measured as change in optical density at 600 nm at the lowestdilution. The growth of control tubes, measured as optical density at600 nm at similar dilution, was considered 100%. The test results wereexpressed as percent viability as compared to the Controls (Table 4).

[0070]Lactobacilli acclimatized to oil environment within the first weekfollowing emulsification. In subsequent weeks, however, the viability oflactobacilli gradually diminished in certain oils i.e. hazelnut,primrose, pumpkin and terila, suggesting a low probiotic recovery. Incontrast, fish oil (omega), olive oil (extra-virgin), rice-bran oil, andsoybean oil all provided an excellent recovery of lactobacilli.Moreover, based on the results expressed in Table 4 below, the presentinventors have reasoned that flax seed oil and vitamin E oil may alsodemonstrate similar prebiotic activity. TABLE 4 Recovery of Lactobacillifrom selected oils in MRS broth (37° C.). Oil-emulsion Type 1^(st) week2^(nd) week 3^(rd) week 4^(th) week Fish (Omega) 122% 220% 255% 280%Hazelnut  92%  88%  67%  59% Olive (Extra virgin) 195% 314% 379% 374%Primrose  87%  87%  94%  95% Pumpkin  79%  78%  82%  85% Rice-bran 108%212% 223% 267% Soybean 112% 217% 274% 290% Terila (pure)  96%  97%  88% 77%

Example 2 Preparation of Probiotic Samples in Nitrogen Purged InstantlyBonded Capsules and Testing

[0071] Each gelatin/vegetable capsule prepared by the NPIB systemcontained about 550 mg emulsion comprising of 109 CFU of lactobacilliand 0.5 ml of fish (Omega), olive, rice-bran or soybean oil. Tencapsules of each oil-emulsified lactobacilli were kept in several glassstorage bottles with air-tight caps and stored at room (28° C.) orrefrigerated (4° C.) temperatures. Bottles of each oil-emulsion typewere taken from both storage conditions, periodically, each month, andthe capsular contents were tested for viability of lactobacilli (colonyforming units, CFU). Capsules made with NPIB system containing 10⁹ CFUlactobacilli without any oil served as controls.

[0072] Each gelatin/vegetable capsule was allowed to dissolve in testtubes containing 10 mL MRS broth for about 30 minutes at roomtemperature. After release of the capsular contents, tubes werethoroughly homogenized using a vortex blender. Serial 10 fold dilutionsof the homogenized mixture were prepared in MRS broth tubes. All tubeswere incubated at 37° C. for 18 h. The growth of control tubes, measuredas optical density at 600 nm at similar dilutions, was considered 100%.Real-time PCR was also used to determine the purity of the revivedstrain KE01 from the encapsulated oil-mixtures and the totallactobacillus mass. The test results were expressed as % viability ascompared to the Controls (Table 5).

[0073] All four types of oil-emulsified probiotic lactobacillidemonstrated a significant recovery rate ranging from 400% to 760% overa period of twelve-month storage at room temperature. These datacompared to the 100% revival rate of control lactobacilli suggested apotent prebiotic effect for these oil-emulsion types. TABLE 5 Percentviability of oil-emulsified lactobacilli (in capsules prepared by theNPIB system) stored at room (28° C.) temperatures for months and revivedin MRS broth (37° C.). Stored at room temp. (28° C.) Oil-emulsion Type2^(nd) 4^(th) 12^(th) (NPIB-System) Month Month 6^(th) Month 9^(th)Month Month Fish (Omega) 565% 568% 572% 525% 482% Olive (Extra-virgin)735% 763% 759% 683% 403% Rice-bran 484% 439% 448% 382% 267% Soybean 411%409% 401% 289% 230%

[0074] Furthermore, the KE01 cells revived from encapsulatedoil-mixtures were tested for purity and probiotic activity compared totheir stock cultures. Lactobacilli revived from the oil-mixtures showeda high degree of purity, with a 100% identity match with strain KE01according to real-time PCR analysis. These revived lactobacillidemonstrated probiotic profiles, i.e. ammonia/sulfide reduction invitro, intestinal Caco-2 cell adhesion, and competitive exclusion ofenteric pathogens from Caco-2 monolayers, similar to their KE01 stockculture. These data suggested that encapsulation of KE01 in specifiedoil mixtures with NPIB system did not affect or alter the probioticactivity of this lactobacillus upon revival over extend periods of time.

[0075] Storage of oil-emulsified probiotic formulations at roomtemperature is user-friendly and commercially preferred. Such storagecondition could free the probiotic product from any refrigeration andwithout compromising viability of the probiotic. Furthermore, theprebiotic enhancement of cellular revival suggested a potent functionalactivity of the oil-emulsified probiotic encapsulations.

[0076] Additional variations considered within the scope of the presentinvention include the addition of viscosity enhancers to theencapsulated contents and extra-capsular coatings. For example,viscosity enhancers such as glycerols (eg. glycerine); glycols (e.g.,polyethylene glycols, propylene glycols); plant-derived waxes (e.g.,carnauba, rice, candililla), non-plant waxes (beeswax); emulsifiers(e.g., lecithin); and silicas (e.g., silicon dioxide) are compatibleingredients for NPIB technology. These viscosity enhancers could provideuniform dispersion of the capsular contents and also could improvemicroaerophilic/anaerobic conditions for the probiotic organism in theencapsulation.

[0077] Extra-capsular coatings serve various applications includingtaste masking, delayed release, vapor/gas diffusion barrier, flavor,color, aroma, enteric protection and leak safety. Such coatings includebut are not limited to celluloses (e.g., methyl- and ethyl-celluloses);methacrylates; and shellacs (e.g., pharmaceutical glazes), just to namea few.

Conclusion

[0078] In closing, it is to be understood that the embodiments of theinvention disclosed herein are illustrative of the principles of thepresent invention. Other modifications that may be employed are withinthe scope of the invention. Thus, by way of example, but not oflimitation, alternative configurations of the present invention may beutilized in accordance with the teachings herein. Accordingly, thepresent invention is not limited to that precisely as shown anddescribed.

[0079] The terms “a” and “an” and “the” and similar referents used inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

[0080] Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

[0081] Preferred embodiments of this invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on those preferred embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

[0082] Furthermore, numerous references have been made to patents andprinted publications throughout this specification. Each of the abovecited references and printed publications are herein individuallyincorporated by reference.

1 2 1 20 DNA Lactobacillus casei 1 ttgtcaccgg cagttcttac 20 2 19 DNALactobacillus casei 2 tgtcgtcagc tcgtgtcgt 19

What is claimed is:
 1. A probiotic composition comprising: a Lactic AcidBacteria (LAB) suspended in an edible oil wherein said edible oil hasprebiotic properties.
 2. The probiotic composition according to claim 1wherein said LAB is selected from the group consisting of Lactobacillusacidophilus, L. amylovorus, L. brevis, L. bulgaricus, L. casei spp.casei, L. casei spp. rhamnosus, L. crispatus, L. delbrueckii ssp.lactis, L. fermentum, L. helvaticus, L. johnsonii, L. paracasei, L.pentosus, L. plantarum, L. reuteri, L. sake, Bifidobacterium animalis,B. bifidum, B. breve, B. infantis, B. longum, Pediococcus acidilactici,Propionibacterium acidipropionici, P. freudenreichii, P. jensenii, P.theonii, Streptococcus cremoris, S. lactis, S. thermophilus andcombinations thereof.
 3. The probiotic composition according to claim 1wherein said prebiotic edible oil is selected from the group consistingof fish oil, olive oil, rice-bran oil, soy oil and combinations thereof.4. The probiotic composition according to claim 1 wherein said LAB is L.casei strain KE01 having ATCC number PTA
 3945. 5. The probioticcomposition of according to any one of claims 1 through 4 furthercomprising a hard two-piece capsule wherein said gelatin/vegetablecapsule is nitrogen purged and instant bonded (NPIB).
 6. A probioticcomposition comprising Lactobacillus casei strain KE01 ATCC numberhaving ATCC number PTA 3945 suspended in an edible oil wherein saidedible oil has prebiotic properties and is selected from the groupconsisting of fish oil, olive oil, rice-bran oil, soy oil andcombinations thereof.
 7. The probiotic composition of according to claim6 further comprising a hard gelatin/vegetable capsule wherein saidgelatin/vegetable capsule is nitrogen purged and instant bonded (NPIB).8. A probiotic composition comprising Lactobacillus casei strain KE01ATCC number having ATCC number PTA 3945 suspended in an edible oilwherein said edible oil has prebiotic properties and is selected fromthe group consisting of fish oil, olive oil, rice-bran oil, soy oil andcombinations thereof.
 9. The probiotic composition of according to claim8 wherein said edible oil is fish oil.
 10. The probiotic composition ofaccording to claim 8 wherein said edible oil is olive oil.
 11. Theprobiotic composition of according to claim 8 wherein said edible oil isrice-bran oil.
 12. The probiotic composition of according to claim 8wherein said edible oil is soy oil.
 13. The probiotic composition ofaccording to any one of claims 8 through 12 further comprising astabilizing ingredient selected from the group consisting of soybeanoligosaccharides, fructooligosaccharides, galactooligosaccharides,galactosyl lactose and palatinose, lactulose, lactitol, xylitol,sorbitol, mannitol, trehalose, glucose, sucrose, fructose, maltose,milk, milk powders, whey, whey protein concentrates, casein, caseinhydrolysates, lactoferrin, lactoperoxidase, lactoglobulins,glycomacropeptides, lacto-saccharides, lacto-lipids.
 14. The probioticcomposition of according to any one of claims 8 through 12 furthercomprising a hard two piece capsule wherein said gelatin/vegetablecapsule is nitrogen purged and instant bonded (NPIB).
 15. A probioticcomposition comprising Lactobacillus casei strain KE01 ATCC numberhaving ATCC number PTA 3945 suspended in prebiotic olive oil and whereinsaid probiotic composition is enclosed in a hard two piece capsule, saidhard two piece capsule being nitrogen purged and instant bonded (NPIB).16. A method for making a probiotic composition comprising: emulsifyinga Lactic Acid Bacteria (LAB) in an edible oil to form an emulsifiedprobiotic, said edible oil having prebiotic properties; filling a hardcapsule body with said emulsified probiotic while said capsule is in thevertical orientation; securing a cap onto said vertically aligned hardcapsule body containing said emulsified probiotic to form a filledcapsule; purging said filed capsule with a Noble Gas; and sealing saidcapsule body to said capsule cap while said capsule body and saidcapsule cap are in said vertically aligned orientation.
 17. The methodaccording to claim 16 wherein the emulsifying step further comprises anedible oil selected from the group consisting of fish oil, olive oil,rice-bran oil, soy oil and combinations thereof.
 18. The methodaccording to claim 16 wherein the emulsifying step further comprises anLAB selected from the group consisting of Lactobacillus acidophilus, L.amylovorus, L. brevis, L. bulgaricus, L. casei spp. casei, L. casei spp.rhamnosus, L. crispatus, L. delbrueckii ssp. lactis, L. fermentum, L.helvaticus, L. johnsonii, L. paracasei, L. pentosus, L. plantarum, L.reuteri, L. sake, Bifidobacterium animalis, B. bifidum, B. breve, B.infantis, B. longum, Pediococcus acidilactici, Propionibacteriumacidipropionici, P. freudenreichii, P. jensenii, P. theonii,Streptococcus cremoris, S. lactis, S. thermophilus and combinationsthereof.
 19. The method according to claim 18 LAB is L. casei strainKE01 having ATCC number PTA
 3945. 20. The method according to claim 16wherein the filling step further comprises a hard capsule derived fromvegetables, animal gelatin synthetic polymers or natural polymers. 21.The method according to claim 16 wherein the purging step furthercomprises a gas selected from the group consisting of Nitrogen, Argonand carbon dioxide.
 22. The method according to claim 16 wherein thesealing step further comprises an organic solvent selected from thegroup consisting of isopropyl alcohol and ethanol.
 23. A pair of KE01specific primers wherein said KE01 specific primers consists of positiveand negative strand and said positive primer is SEQ ID NO 1 and saidnegative strand in SEQ ID NO 2.